Rheumatoid arthritis (RA), the most common inflammatory joint disease of autoimmune etiology, affects nearly 1% of the population. It is clear that the adaptive and innate immune systems are important in the pathogenesis of RA. Autoantibodies that target citrullinated proteins are specific to RA, and innate immune receptors such as Toll-like receptors (TLRs) play important roles in the induction of both inflammation and adaptive immunity (e.g. B- and T-cell activation). A recently identified target of the anti-citrullinated protein antibody (ACPA) response in RA is citrullinated fibrinogen (cFb). We demonstrated that immune complexes (ICs) containing cFb (cFb-IC) are present in the blood and synovial tissue of patients with RA, and that immunization of mice with cFb induces inflammatory arthritis. Why cFb-specific autoimmunity should mediate inflammatory arthritis remains unclear. Investigating the mechanisms by which immunization with cFb evokes inflammatory arthritis, we observed that cFb itself is a powerful innate adjuvant that signals through TLR4, and that cFb-IC can synergize to augment macrophage TNF production by co-ligating TLR4 and the Fc3 receptor (Fc3R). Our preliminary studies also indicate that cFb can stimulate proliferation of B cells in a TLR4- dependent manner. In addition, we identified significantly elevated levels of plasmablasts-the early, activated B cells-in the blood of ACPA-positive RA patients. These circulating plasmablasts produce large amounts of ACPA, including anti-cFb antibodies, suggesting that ACPA-positive RA is associated with ongoing, antigen- driven activation of peripheral B cells. Finally, we detected the presence of citrullinated proteins, including citrullinated fibrinogen, not only in the inflamed joint, but also within the atherosclerotic plaque of RA and non- RA subjects. Our overriding hypothesis is that inflammation in RA is mediated by cFb-IC, which drive proinflammatory responses by augmenting macrophage TNF production, and adaptive immune responses by activating B cells specific for cFb;and that the innate and adaptive immune response induced by cFb-IC together propagate the synovial and extra-articular manifestations of RA. We aim to demonstrate 1) that antigen-driven B-cell activation results in plasmablast formation, and that these plasmablasts are a major source of ACPA and 2) that TLR4 is upregulated on B cells at sites of chronic inflammation and that cFb-IC can co-stimulate these B cells via co-ligation of TLR4 and the B-cell receptor. To achieve these aims we will use novel and established models of immune complex arthritis, in vitro cell culture and flow-cytometric analysis,and a novel bead-based antigen microarrayfor profiling of RA patients'autoantibodies as well as antibodies produced by cultured plasmablast. Success of these studies will provide insight into the pathways mediating inflammation in RA, as well as further evidence of autoantigen-driven clonal expansion of self- reactive B cells. Proinflammatory cytokines and B cells are both targeted by current RA therapies, and an improved understanding of the mechanisms of innate and adaptive immune responses in RA could yield new and potentially better targets for disease prognostication as well as therapeutic intervention. PUBLIC HEALTH RELEVANCE: Rheumatoid arthritis (RA) is a chronic autoimmune arthritis that affects nearly 1% of the United States population and is a leading cause of disability and health-care expenditures among veterans. Success of the proposed experiments would demonstrate that citrullination-a specific modification of the body's proteins- and the development of autoantibodies that target citrullinated proteins are crucial not only to morbidity caused by inflammation of the joints, but potentially also to the increased cardiovascular mortality associated with RA. The proposed experiments will provide insights into diseaes pathogenesis that could lead to development of new therapeutic approaches for RA and its associated disability.